Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity

Summary Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.


INTRODUCTION
Cystic fibrosis (CF) is a life-limiting genetic disease resulting from mutations in the CF transmembrane conductance regulator (CFTR) gene (Ratjen et al., 2015). CFTR-the only member of the ABC transporter family known to be an ion channel-consists of two transmembrane domains (TMD1 and TMD2) which form an anion-selective pore, two highly conserved nucleotide-binding domains (NBD1 and NBD2) with ATPbinding pockets and a newly described N-terminal lasso motif (Hwang and Kirk, 2013;Zhang and Chen, 2016). In addition, CFTR has a unique, disordered regulatory (R) domain which contains protein kinase A (PKA) phosphorylation sites. For the CFTR channel to open and close (gate), cAMP-dependent PKA phosphorylation of the R domain first activates the CFTR (Gadsby and Nairn, 1994). Then, ATP-binding induces the dimerization of the two NBDs which opens the channel pore and ATP hydrolysis closes the pore.
CFTR modulators, small molecules which directly target CFTR dysfunction, are now available to certain individuals with CF. Currently, two classes are approved; (1) potentiators, which open the channel pore such as ivacaftor  and (2) correctors, which assist CFTR protein folding and delivery to the cell membrane. Type I correctors (lumacaftor/VX-809, tezacaftor/VX-661) stabilize the NBD1-TMD1 and/or NBD1-TMD2 interface by binding directly to TMD1 (Loo et al., 2013;Ren et al., 2013) or NBD1 which improves the interaction between NBD1 and the intracellular loops (Hudson et al., 2017;Loo and Clarke, 2017). Type II correctors (C4) stabilize NBD2 and its interface with other CFTR domains while type III correctors (elexacaftor/VX-445) directly stabilize NBD1 (Okiyoneda et al., 2013). Combination therapies of corrector(s) and a potentiator (Orkambiâ, Symdeko/Symkeviâ, Trikafta/Kaftrioâ) have been approved for CF individuals with F508del, the most common CFTR mutation, as well as several specific residual function mutations. Most recently, Trikafta/Kaftrio has been approved for patients with a single F508del mutation in combination with a minimal function mutation, broadening the population of patients with CF eligible for treatment with CFTR modulator therapy.
Mounting evidence has shown that in vitro functional studies in patient-derived cell models successfully predict clinical benefit of available CFTR modulators for individuals bearing ultra-rare mutations (Berkers et al., 2019;McCarthy et al., 2018;Ramalho et al., 2021). In individuals with CF, adult stem cells are usually collected by taking either airway brushings or rectal biopsies. Single Lgr5 + stem cells, derived from crypts within a patient's intestinal epithelium, can be expanded in culture medium and differentiated into organized multicellular structures complete with the donor patient's genetic mutation(s), thus representing the individual patient (Sato et al., 2009). Stem cell models can be used for personalized drug screening to theratype and characterize rare CFTR mutations (Awatade et al., 2018;Berkers et al., 2019;Pollard and Pollard, 2018). Determining the functional response of rare, uncharacterized CFTR mutations to modulator agents with known CFTR correction mechanisms enables characterization of CFTR structural defects and enhances our understanding of CFTR function.
I37R-CFTR is a novel missense mutation in the lasso motif, detected in an Australian male child diagnosed through newborn screening with elevated immunoreactive trypsinogen, raised sweat chloride (>60 mmol/ L), and CFTR Sanger sequencing identifying c.1521-1523del (F508del) and c.110C > T (I37R) mutations (Table S2). We used functional studies and molecular dynamics (MD) simulations to characterize the functional and structural defects of I37R-CFTR. CFTR function was assessed using intestinal current measurements (ICM) in rectal biopsies, forskolin-induced swelling (FIS) assays in intestinal organoids, and short circuit current measurements (I sc ) in I37R/F508del organoid-derived monolayers, respectively. The potentiators VX-770 (approved), GLPG1837 (phase II clinical trials), and genistein (a natural food component with potentiator activity (Dey et al., 2016)) were tested as monotherapies, dual potentiator therapies, or in combination with correctors . We compared this to our laboratory reference intestinal organoids. For MD simulations, we modeled and examined the structural defect of the I37R mutation on an extended cryo-EM structure of ATP-bound, phosphorylated human CFTR (PDB ID code 6MSM) (Zhang et al., 2018).
Co-activation with carbachol (CCh) resulted in a biphasic response in the I37R/F508del biopsies, characteristic of residual CFTR chloride channel function in the CF colon (Graeber et al., 2015;Veeze et al., 1994). The initial negative I sc peak indicates apical potassium secretion reached 9.4 G 2.5 mA/cm 2 . Following this, the CCh-induced positive I sc indicates the increase of apical chloride secretion reached 15.78 G 2.07 mA/cm 2 . This biphasic response was similarly observed in the G551D/F508del biopsies (25.77 G 2.16 mA/cm 2 ) but was diminished in the F508del/F508del biopsies (À2.28 G 1.65 mA/cm 2 ). These findings are in accordance with the localization of CFTR protein at the plasma membrane (mature complex-glycosylated CFTR) of the I37R/F508del rectal biopsies, as demonstrated by immunofluorescence staining (green; Figure 1B). (D)Western blot in WT/WT, F508del/F508del, and I37R/F508del intestinal organoids. CFTR maturation was calculated by measuring the level of mature mutant CFTR (Band C) as a percentage of mature CFTR from WT organoids (% normal CFTR). All data were normalized to the calnexin loading control. B and C represents the mature, complex-glycosylated CFTR. B and B represents the immature, core-glycosylated CFTR. See Figure S9 for uncropped Western blot images. (E and F) Forskolin-induced swelling (FIS) assay in organoids from participants with F508del/F508del (n = 5), G551D/F508del (n = 2), and I37R/F508del (n = 1) CFTR genotypes. Organoids were stimulated with forskolin (fsk) concentrations ranging from 0.02 to 5 mM.(E) FIS expressed as the means G standard deviation (SD) of the area under the curve (AUC) calculated from t = 0 (baseline) to t = 60.(F) FIS of organoids at 0.8mM fsk at baseline represent residual CFTR function. Data represented as violin plots with mean to show distribution. (G) Immunofluorescence staining of e-cadherin (green), ZO-1 (red), and DAPI (blue) in organoid-derived monolayers from a CF participant. 20x/0.75 dry objective. Scale bars = 50 mm. (H) Representative Ussing chamber recordings of short circuit current in organoid-derived monolayers from a WT-CFTR control participant and participants with CF. Dot plots of fsk-induced current (DIsc-Fsk) in participants with WT/WT (n = 1), F508del/F508del (n = 1), and I37R/F508del (n = 1) CFTR genotypes.
Given the I37R/F508del high residual CFTR activity and its localization at the epithelial cell surface, we hypothesized that the I37R-CFTR mutation has minimal impact on CFTR protein folding or maturation. Treatment of I37R/F508del organoids with type I corrector agents (VX-809 or VX-661) did not significantly increase FIS above baseline (Figures 2E-2G and S1). In contrast, treatment of I37R/F508del organoids with a type III corrector agent (VX-445) significantly increased FIS by AUC of 1112.5 above baseline, greater than those in the F508del/F508del organoids (42.5). VX-445 has been shown to act as both a corrector and potentiator for certain CFTR mutations (Laselva et al., 2021;Shaughnessy et al., 2021;Veit et al., 2021a). Acute treatment of I37R/F508del organoids with VX-445 did not improve potentiation of CFTR ( Figure S1). This supports the observation that VX-445-stimulated rescue of CFTR in I37R/F508del organoids acts by a correction mechanism improving I37R mild folding and processing defects. I37R-CFTR functional response to CFTR modulator co-therapies in intestinal organoids.
Combination treatments of CFTR modulators are used to treat patients bearing CFTR mutations with multiple functional defects such as F508del and patients who are heterozygous for CFTR mutations. We investigated the effect of combinations of potentiators. Dual potentiator combinations increased FIS of I37R/ F508del organoids to a greater extent than the respective single potentiators ( Figure 3A) and had a synergistic effect, where the FIS was greater than the sum of the respective single potentiators (Table S4). iScience Article Despite G1837 + Gen having greater efficacy than the other dual potentiator combinations, the magnitude of response was not statistically different between the different combinations of dual potentiators ( Figure 3A).
Co-therapy with a corrector (VX-809 or VX-661) and dual potentiators significantly (p < 0.01) increased FIS of I37R/F508del organoids compared to co-therapy of a corrector with VX-770 or Gen, but not G1837 (G) FIS of organoids at 0.128uM fsk following incubation with corrector (VX-809 or VX-661 or VX-445) for 24 h. Data corrected for baseline FIS and represented as violin plots with mean to show distribution. One-way analysis of variance (ANOVA) was used to determine statistical differences except in (D) where unpaired t test was used. **p < 0.01, ***p < 0.001, and ****p < 0.0001. aP for G1837, bP for Gen and cP for VX-445 of I37R/F508del,^P for G1837 vs VX-770, or Gen and #P for VX-445 vs VX-809 or VX-661.
Dual correctors (VX-445+VX-661) increased FIS in I37R/F508del organoids by AUC of 1856.6 above baseline, which corresponds with the level of rescue achieved by the most effective corrector/dual potentiator co-therapy (VX-809/G1837 + Gen). The triple combination therapy with dual correctors and a potentiator further increased FIS in I37R/F508del organoids by AUC of 3101.6 above baseline. It is therefore the most effective modulator combination tested in this study.

I37R-CFTR perturbs the noose structure of the lasso motif
We next characterized the structural defect of I37R-CFTR using MD simulations. The primary structure of the lasso motif (M1-L69) is conserved across 230 vertebrate species ( Figure S2, Table S5). The lasso motif formed a noose structure that rested against TMD2 ( Figure 4A). Amino acids V12-R29 were embedded in the plasma membrane while the rest of the lasso motif resided in the cytosol. The noose structure was maintained by a salt bridge formed between K26 and D36 ( Figure 4B). I37 was positioned in the center of this noose, within a hydrophobic pocket formed by amino acids from the lasso, TMD2, and the poorly resolved R domain in the cytosol ( Figure 4C).
Mutation of the evolutionarily conserved, non-polar and uncharged isoleucine (I) of I37 to a positively charged arginine (R) introduced an unstable lone charge into the hydrophobic pocket within the lasso motif noose. We hypothesized that this likely results in the rotation of the R37 side chain out of the hydrophobic pocket, and possible coordination with negative charges in the nearby R domain.
To identify a reasonable conformation of the mutant lasso motif, the WT 6MSM model was mutated to R37 and three 2 ms simulations were performed at physiological temperature (310 K). The R37 side chain rotated iScience Article out of the hydrophobic pocket in only one of the three simulations. The difference between the root-meansquare deviation (RMSD) of the noose structure of I37R-CFTR compared to the WT was on average 2.8 Å at the amino acids M1-L6, and 1.8 Å at L34-S50 ( Figure 4D). To confirm this observation, repeat simulations were performed at 350 K (40 above physiological temperature), a temperature shown to accelerate the potential conformational transitions of proteins (Beckerman, 2015). In these higher temperature simulations, the root-mean-square fluctuation (RMSF) of the region around amino acid 37 doubled in two out of three simulations, compared to WT-CFTR at 310K ( Figure S3). This confirmed the destabilization of the lasso motif by I37R-CFTR. All WT-CFTR domains and the surrounding bilayer remained stable at the elevated temperature ( Figures S4 and S5).

I37R mutation strengthens lasso motif interaction with the R domain
In the 6MSM structure, the R domain is largely unresolved with two exceptions: the first (Q637) and last (T845) amino acids that adjoin neighboring domains, and the backbone atoms of a 17 amino acid segment. This latter segment consists of an eight amino acid disordered coil followed by a nine amino acid alphahelix (Zhang et al., 2018). The alpha-helix was separated by approximately 10 Å (1 nm) minimum C-alpha distance to I37 in the lasso motif. This suggested a likely interaction between this segment of the R domain and I37, which necessitated partial modeling of the R domain ( Figure 5A).
Modeling of these 17 unidentified amino acids was performed by creating 24 different in silico models of this segment based on the 6MSM structure. In each model, a unique 17 amino acid sequence was determined with a sliding window of one amino acid, starting backwards from amino acid T842 due to the  iScience Article alpha-helix's 20 Å proximity to T845. The 17 amino acids were then connected to T845 with the missing linking amino acids. The structural stability of all 24 modeled segments was tested by performing up to 300 ns simulations for each model and comparing the backbone RMSD measurements against 6MSM ( Figures 5B  and S6). The model with the lowest RMSD (3 Å ) and thus the highest stability was attained when L818-F834 was assigned to the unidentified 17 amino acids, of which the alpha-helix maps to E826-F834 ( Figures 5B  and S6). This assignment was corroborated by NMR measurements of the isolated R domain in solution, where the same segment retained partial helicity (Baker et al., 2007). Predictions of the structure of human CFTR by Alphafold2 also aligned with this assignment of primary structure to the unidentified amino acids ( Figure S7) (Jumper et al., 2021). Several favorable interactions between this R domain model and other parts of the CFTR protein further supported this assignment ( Figures 4C and 5D). Two hydrophobic amino acids (L829 and F833) contributed to the hydrophobic pocket that stabilized the lasso motif around I37. The negatively charged E831 formed a salt bridge with positively charged K968 in TMD2. Together, these interactions secured the R domain alpha-helix into position throughout an extended 2 ms simulation, resulting in a smaller minimum C-alpha distance to the lasso motif of 8.9 G 0.2 Å compared to the 10 Å in the 6MSM cryo-EM structure.
The reoriented R in position 37 in the I37R mutant protein, which pointed out of the hydrophobic pocket, rearranged the salt bridge network supporting the lasso motif by breaking the evolutionarily conserved salt bridge K26-D36. Two new salt bridges were formed, one with the negatively charged E823 and another with E826 of the R domain ( Figure 5C). Furthermore, the E831-K968 salt bridge between the R and TMD2 domains in the WT was exchanged for a D828-K1080 salt bridge in I37R-CFTR ( Figure 5C). The  iScience Article backbone motions required to accommodate these new charge interactions also perturbed parts of the lasso motif ( Figure S3) and R domain. The lasso N-terminus shifted its position towards the R domain and reduced the minimum C-alpha distance between them by 3.5 Å ( Figure 5D). The overall result was a tighter coupling between the lasso and the R domain which is anticipated to inhibit the R domain movements required for channel gating.

DISCUSSION
We have described the functional and structural defects of I37R, a novel CF-causing mutation in the segment of the CFTR lasso motif which interacts with the R domain. These were compared to reference CFTR mutations which have known functional defects, either a CFTR folding/maturation (F508del/ F508del) or a gating (G551D/F508del) defect. First, ICM performed in I37R/F508del rectal biopsies identified I37R confers high residual activity (50% of WT-CFTR activity). High baseline CFTR activity was similarly observed in FIS of I37R/F508del intestinal organoids and I sc measurements in organoid-derived monolayers. Given we and others showed that F508del is a severe mutation which contributes little functional CFTR (Van Goor et al., 2011), this suggests that I37R mutation produces CFTR protein which localizes to the epithelial cell surface. These observations are consistent with the patient's mild CF clinical phenotypes (pancreatic sufficient with faecal elastase>500 mg/g, FEV 1 z-score -0.11, 99% predicted).
We also characterized the response of I37R-CFTR to modulators (potentiators and correctors) in I37R/ F508del intestinal organoids and organoid-derived monolayers. I37R was responsive to potentiators which improve CFTR gating function and a newly approved corrector (VX-445). Among the three potentiator agents tested, the response to VX-770 was minimal. The reason for the lack of efficacy of VX-770 is not known, because molecular modeling studies propose that VX-770 shares the same mechanism of action and binding sites with G1837 (Liu et al., 2019;Yeh et al., 2019). Both VX-770 and G1837 are proposed to potentiate CFTR by increasing channel open probability (Po) through stabilization of the open-pore conformation, independent of NBD dimerization and ATP hydrolysis which normally controls channel gating (Van Goor et al., 2009;Yeh et al., 2017). However, the differing potentiator efficacies are not a new observation. G1837 was previously shown to be more potent and effective than VX-770 in human bronchial epithelial cells from a G551D/F508del and a R334W/F508del CF participant (Gees et al., 2018;Van der Plas et al., 2018). Similar observations were reported in heterologous HEK293 cells expressing Class III (G551D, G178R, and S549N) and Class IV (R117H) CFTR mutants (Gees et al., 2018;Van der Plas et al., 2018). We conclude that perhaps G1837 has additional binding sites or actions distinct from VX-770, which in the case of I37R-CFTR, results in significant potentiation of the CFTR channel.
We further showed that dual potentiator combinations exerted synergistic restoration of CFTR activity in I37R/F508del organoids. This synergistic restoration is not exclusive to I37R-CFTR, because similar findings have been reported for other CFTR mutations responsive to potentiators (Dekkers et al., 2016b;Phuan et al., 2018Phuan et al., , 2019Veit et al., 2019). Synergism is commonly achieved when potentiators have distinct binding sites and mechanisms of actions. One potentiator could induce allosteric interactions that favor the activity of the other potentiator (Nussinov and Tsai, 2013). The potentiator synergy observed in our dual potentiator combinations supports our hypothesis that G1837 may have additional binding sites or mechanisms of action to VX-770. While VX-770 has been shown to provide clinical benefit to patients with responsive mutations (Berkers et al., 2020;McKone et al., 2014;Volkova et al., 2020), it does not restore the Po of gating defect mutants (G551D-CFTR) to full WT-CFTR activity (Van Goor et al., 2009). This opens the possibility that using another potentiator with a different mechanism of action could complement VX-770 activity and increase CFTR activity beyond that of VX-770 monotherapy. While VX-770 and G1837 act independently of NBD dimerization and ATP hydrolysis (Van Goor et al., 2009;Yeh et al., 2017), genistein promotes ATP-dependent gating of CFTR by binding to the NBD1/2 interface and inhibiting ATP hydrolysis (Sohma et al., 2013). Genistein has been demonstrated to increase VX-770-potentiated CFTR activity in intestinal organoids, even when VX-770 was used at near-saturating concentrations (Dekkers et al., 2016b). Our observations reiterate and expand on these findings to suggest that potentiators with different mechanisms of action could provide synergistic restoration of CFTR activity to responsive CFTR mutations compared to potentiator monotherapy.
Chronic treatment with type III corrector VX-445 rescued CFTR activity in I37R/F508del organoids, while neither type I correctors (VX-809 or VX-661) rescued activity. This response is attributed to the I37R and not the F508del mutation in the I37R/F508del organoids, because VX-445 did not restore CFTR activity ll OPEN ACCESS iScience 25, 103710, January 21, 2022 9 iScience Article in F508del/F508del organoids. While VX-445 has been shown to have partial potentiator activity (Laselva et al., 2021;Shaughnessy et al., 2021;Veit et al., 2021a), VX-445 did not potentiate CFTR activity in I37R/ F508del organoids when administered acutely. This is the first study to interrogate the potentiator action of VX-445 in intestinal organoids; however, previous studies have been performed in donor-derived bronchial and nasal epithelial cells and immortalized cell lines. The higher correction efficacy of VX-445 when compared with VX-809/VX-661 has previously been shown, although this is likely to be dependent on the CFTR variant (Keating et al., 2018;Veit et al., 2020Veit et al., , 2021b. For instance, direct binding of VX-445 to NBD1 to stabilize and prevent the domain unfolding may make it more effective in correcting CFTR mutations that impact NBD1 function (such as F508del located in NBD1).
The lack of I37R-CFTR correction by VX-809 or VX-661 could be attributed to the dependency of these modulators binding to and stabilizing the TMD1. TMD1 function is modulated by interaction with lasso helix 2 (Lh2, aa A46-L61) as deletion of Lh2 from the WT CFTR was shown to completely abrogate VX-809-mediated CFTR maturation (Sabusap et al., 2021). MD studies showed that VX-809 occupancy at the TMD1 binding site causes the Lh2 to move, such that the network of salt bridges in Lh2 holds TMD1 (CL1) and TMD2 (CL4) in the correct orientation (Baatallah et al., 2021;Okiyoneda et al., 2013). This then allows for allosteric coupling between NBD1 and TMD1 or 2, which is important for cooperative domain folding of CFTR. In support of this, mutation of critical amino acids at the binding pocket of VX-809 on CFTR, or those involved in the architecture of this site, were shown to diminish the sensitivity to VX-809 correction. L53V and F87L mutations, which are located in the vicinity of the VX-809 binding site in the TMD1, were shown to prevent VX-809 correction in F508del HEK283 cells (Baatallah et al., 2021). Considering the above and because I37 is only a few amino acids away from the Lh2, it is plausible that the local conformational changes associated with the I37R mutation which we have identified in our study ( Figure 4D) may disrupt the allosteric coupling between NBD1 and TMD1 or 2, preventing correction with type I correctors.
CFTR missense mutations in the lasso motif are not well characterized. This is because most of these mutations are rare, with an allele frequency of less than 0.01% in the CF population (Table S1). The only characterized missense mutations in the region of the lasso motif where I37 resides-between Lh1 (amino acid 19-29) and Lh2 (amino acid 46-61)-are R31C and R31L (CFTR2, 2021;Jurkuvenaite et al., 2006). Experimental studies in heterologous COS-7 cells showed both mutations cause a mild processing defect and accelerated CFTR internalization. Individuals heterozygous for these CFTR mutations are reported to have a mild disease phenotype with pancreatic sufficiency (Jurkuvenaite et al., 2006). One individual with the R31C/F508del CFTR genotype was reported to have a normal sweat chloride level (25 mmol/L) and nasal potential difference (Werlin et al., 2015). CFTR2 classifies R31C as a non-CF disease causing mutation. Notably, mild disease phenotypes (mild pulmonary symptoms, pancreatic sufficiency) are reported for several other lasso motif missense mutations including P5L, E56K, and P67L (Table S1), as was found for the I37R/F508del participant in this study. This suggests that perhaps lasso motif mutations do not significantly impact the overall CFTR structure and function given its short length (69 of 1480 amino acids, 4.7%). It is also plausible that the role of the lasso motif could be compensated for by other CFTR domains.
To better understand the functional defect of I37R-CFTR, we used MD simulations to model the structural features of I37R and how they are altered relative to WT-CFTR. The amino acids 34-39 were shown to interact with the R domain in the phosphorylated, ATP-bound CFTR structure (Zhang et al., 2018). This interaction was absent in the closed conformation of CFTR (Zhang and Chen, 2016), suggesting that the short region of amino acids 34-39 interacts with the R domain to regulate CFTR channel gating. We found that the disruption of the evolutionarily conserved K26-D36 salt bridge in I37R-CFTR brings the lasso motif closer to the R domain. We also found that the I37R side chain rotates out of its hydrophobic pocket to form interactions with negatively charged E823 and E826 on the R domain. We speculate that R37 clamps the lasso motif to the R domain, preventing the dynamic movement of the two domains necessary for a normal CFTR opening and closing cycle, thus causing a gating defect. This supports our functional observations, wherein I37R-CFTR demonstrated significant responsiveness to potentiator agents which are known to increase channel opening time. Furthermore, in the I37R-CFTR model, conformational changes in the lasso motif were also evident but were limited to short regions (M1-L6, L34-S50), indicating that the overall architecture of the CFTR protein remains largely intact. Additionally, our simulations did not show any change to the pore architecture of CFTR ( Figure S8).
The simulated structure in this work is of CFTR in its active state (Zhang et al., 2018). Because of this, we believe the pathogenic interactions discovered in this study have a significant contribution to the ll OPEN ACCESS iScience 25, 103710, January 21, 2022 iScience Article deleterious effects of the I37R mutation. However, the enhanced lasso motif-R domain interactions should be interpreted in the context of the ms timescales reachable by unbiased simulations. The lasso domain is known to exhibit conformational flexibility during both folding and functional stages of CFTR (Kleizen et al., 2021), which take place on timescales longer than is currently feasible to study in atomistic simulations. Therefore, there may be pathogenic interactions in I37R-CFTR in addition to the ones captured by the simulation of this particular CFTR structure.
The I37R/F508del participant in this study will only meet the Therapeutic Goods Administration (Australia) requirements for treatment with Trikafta/Kaftrio triple combination therapy once he turns 12 years old given the single copy of the F508del mutation. He is not eligible for single potentiator therapy or corrector/potentiator combinations of lumacaftor/ivacaftor or tezacaftor/ivacaftor. This emphasizes the importance of characterizing the structural and functional defects of ultra-rare CFTR mutations together with the assessment of in vitro response to modulator drugs in patient-derived cell models to build the case for access to treatment with available modulators through precision medicine health technology assessment pathways. Furthermore, when multiple CFTR modulators are available to patients with CF, determining the best modulator for patients with a rare mutation not investigated in a clinical trial may be supported using in vitro personalized cell models.

Limitations of the study
Organoids often lack specialized cell types and fail to recapitulate the complexity of native organs (Clevers, 2016). For example, mesenchymal, endothelial, and microbiome are absent from intestinal organoids. Integration of such features remains technically challenging and their absence may impact drug response. Another important drawback of organoid systems is the heterogeneity in their size when seeded for FIS assay. As the size of organoids increases, diffusion-dependent drug supply becomes less efficient. This may in turn impact the accuracy of outcome of drug assay. Reducing this variability will be essential to fully capitalize on the potential of organoids in drug screening. Another limitation of the organoid systems is the variability in the magnitude of FIS response in intestinal organoids across different CF laboratories. This is due to the dependence of organoids on media that is developed inhouse with many locally produced media factors Ramalho et al., 2021). This limitation can be resolved by the creation of reference donor organoids which are made available and used internationally between CF laboratories.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr Shafagh Waters (shafagh.waters@unsw.edu.au).

Materials availability
This study did not generate new unique reagent.

Data and code availability
MD simulations data of an equilibrated model of CFTR with the missing R domain fragment has been deposited at Zenodo and is publicly available as of the date of publication. DOI is listed in the key resources table. All other data reported in this paper will be shared by the lead contact upon request.
All original code for MD studies has been deposited at Zenodo and is publicly available as of the date of publication. DOI is listed in the key resources table. The code for FIS assay will be shared by the lead contact upon request.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Quantification of forskolin-induced swelling
Organoid swelling was quantified using a custom-built script. A segmentation strategy implemented using ImageJ/Fiji was performed on brightfield images. The raw image was processed with a gaussian blur (s=1.3) to reduce noise. After the directionality and magnitude of the local gradient was identified, pixels were classified as either 'Background', 'Ridge', 'Valley', 'Rising' or 'Falling' dependent on their neighbouring pixels along the previously calculated local directionality. Clean-up filters were applied that remove noise and small objects, such as ridges that only touched background pixels, and erosions to decrease rising and falling edges to better approximate object boundaries ('Peaks'). A size exclusion was applied that would discriminate debris in the sample preparation from organoids of interest. This segmentation strategy was used to identify area covered by organoid at each time point. The total surface area of organoid at 10-min intervals over 60 min post-fsk stimulation were calculated and normalized against t=0 to render the relative amount of swelling from t=0. The area under the curve, AUC (calculated increase in organoid surface area from t=0 to t=60; baseline=100%) was then calculated using GraphPad Prism software.